PROJECT SUMMARY/ABSTRACT Megakaryocytes (MK) are rare very large marrow cells that give rise to blood platelets. Recent evidence has implicated MKs in regulating hematopoietic stem/progenitor cell (HSPC) activity by the many cytokines and extracellular matrix components produced by these cells. Many hematologic malignancies are associated with MK abnormalities. The myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by HSPC expansion and overproduction of mature blood cells. The acquired mutation JAK2V617F plays a central role in these disorders, but the precise molecular mechanisms responsible for MPN HSPC expansion are not fully understood, limiting the effectiveness of current treatments. MK hyperplasia is a hallmark feature of MPNs. We have shown that JAK2V617F-bearing MKs cause a murine myeloproliferative syndrome with HSPC expansion and increased marrow sinusoidal vascular density. In addition, we have demonstrated that the MPN vascular niche is important for JAK2V617F HSPC clonal expansion. Based on these initial results, we hypothesize that JAK2V617F-bearing MKs have altered hematopoietic niche function which not only enhances stem cell function directly but also affects the vascular niche to indirectly promote HSPC expansion in MPNs. The objective of the proposed work is to determine the physiological effects and the molecular mechanism(s) by which JAK2V617F-bearing MKs maintain and expand HSPCs in MPNs. In particular, we propose the following three specific aims: 1) To study the effects of JAK2V617F-bearing MKs on stem cell function using both a transgenic murine model and human induced pluripotent stem cell lines; 2) To study the effects of JAK2V617F MKs on endothelial cell function in vitro, and the vascular niche and HSPC expansion in vivo. The role of thrombopoietin/MPL signaling in JAK2V617F MK niche function will be assessed; 3) To systemically investigate how JAK2V617F mutation affects megakaryocyte intracellular, membrane, and secreted proteins by quantitative proteomics. The function of selected proteins in HSPC function and vascular niche function will be explored. The long term goal of this research proposal is to identify the complex network of MK signaling in both normal and neoplastic hematopoiesis and develop additional, more effective therapeutic strategies in MPNs and potentially other hematologic malignancies.